Deja Cunningham, Week 2, H&E Staining and Lung Dissection






My second week at Duke has been even more exciting! Early in the week, I learned about two projects I will be working on with the MD/PHD student Elizabeth. The first is an “in vivo” experiment (in the mice) in which we are following the murine mouse model in order to study Sarcoidosis, an autoimmune disease. Sarcoidosis is one of many granulomatous diseases that results in the formation of granulomas, masses of immune cells, in different organs. We are specifically focusing on its formation in the lungs. Through different studies is has been shown that treatment of the lungs with carbon nanotubes (CNTs) increases formation of the granulomas in mice after 60 and 90 days. Oesteopontin, a protein involved in extracellular matrix remolding and formation of the granulomas, also increases with the treatment of CNTs  Elizabeth and I are looking to compare granuloma formation in naïve wild type mice (untreated), osteopontin knockout mice (treated), wild type mice (treated), and naïve osteopontin knockout mice (untreated). This week I took slides, which contained lungs embedded in paraffin and performed a test run of the H&E staining protocol. This method is used to stain the lungs in order to see the granulomas under a microscope.


     Test paraffin embedded lungs treated     
          with Carbon Nanotubes        




  Lung granuloma under microscope after H&E staining
                                                
The second project is an “ex vivo” experiment (out the mice) related to Sarcoidosis. We will be creating ample amounts of macrophages derived from the bone marrow (BMDMs) on petri dishes with the growth factor M-CSF. Once we have a lot  macrophages, they will be treated with different concentrations of the CNTs in order to see how concentration affects granuloma formation. In the future, we will perform RNA purification to see what genes are highly expressed and we will use ELISA, which calculates the amount of protein present, to see osteopontin levels. Later in the week, I helped Elizabeth dissect 11 mice in order to collect the lungs. The lungs will be sent to a CORE facility in Duke where they will be embedded in paraffin. Once we receive the embedded lungs and Elizabeth slices them, I will begin the staining process again.


        Wild type BMDMs treated with CNTs      
                  

Dissected parts of the right lung
                  
On Wednesday and Thursday Dr. Venanzi (and Annamarie!!) visited us. She came to each of our labs and then took us out to dinner Wednesday night at Mother and Sons Trattoria. On Saturday Keeley, my mom, and I visited Elon University and we had lunch at a taco place in town called Tangent, which has THE BEST spicy fried catfish taco ever. Like seriously. We said we would go back just for the tacos. For the rest of the weekend we relaxed to prepare for the upcoming week. So far, this experience has been amazing and I can’t wait to see what week 3 beholds!










                                                                                                                                                                                                                                     

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